1. Field of the Invention
This invention relates to positively charged non-natural amino acids, methods of making thereof, and utilization thereof in peptides. In one embodiment, the invention relates to non-natural amino acids that closely replicate the natural amino acids lysine and arginine.
2. Background
Synthetic studies have been directed toward understanding the influence that only some non-natural amino acids have on the structural and biological activity of peptides.
U.S. Pat. No. 3,178,472 to Hellerbach et al. discloses a method for the conversion of amino carboxylic acids to their N-monomethyl derivatives. Naturally occurring amino acids such as alanine, phenylalanine, serine, cysteine, cystine, tyrosine, tryptophan, histidine, methionine, valine, norvaline, leucine, isoleucine, arginine, ornithine, lysine, aspartic acid, glutarninic acid, threonine, xcex1,xcex3-diaminobutyric acid and the like are suitable starting materials. When the starting material aminocarboxylic acid contains two amino groups such as lysine, omithine or xcex1,xcex3-diaminobutyric acid, then it is possible to generate a product in which one or both amino groups are methylated.
Moore et al. (Can. J. Biochem. 1978, 56, 315) discloses the effect of the basic amino acid side chain length and the penultimate residue on the hydrolysis of benzoyldipeptides by carboxylicpeptidase B1 (CPB). Non-natural amino acids including homolysine and homoarginine were incorporated into small peptide chains, and the kinetic prameters were determined for the CPB catalyzed hydrolysis of the peptide.
Lindeberg et al. (Int. J. Peptide Protein Res. 1977, 10, 240) discloses the synthesis of 1-deamino-4-L-valine-8-DL-homolysine-vasopressin and protected 1-deamino-4-L-valine-8-D-lysine-vassopressin in which with non-natural amino acids were incorporated. The addition of a methylene group to lysine and arginine to generate the non-natural amino acids homolysine and homoarginine, respectively. The study revealed that peptides with homolysine and homoarginine reduced the antidiuretic activity of the peptides.
Hilpert et al. (J. Med. Chem. 1994, 37, 3889) discloses screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of the arginine mimetic (aminoiminomethyl)piperidine as a weak thrombin inhibitor. A number of derivatives of the arginine mimetic were prepared, and their ability to inhibit thrombin was assayed. The X-ray crystal structure analysis of thrombin as well as modeling studies of the arginine mimetic were conducted in order to rationalize the observed affinity between the unnatural amino acid and thrombin.
Nestor et al. (J. Med. Chem. 1988, 31, 65) discloses the synthesis of a new series of unnatural amino acids and their incorporation into antagonistic analogues of lutenizing hormone-releasing hormone (LH-RH). In particular, non-natural amino acids of arginine exhibited high acute potency and very prolonged duration of action. Biological and clinical pharmacology studies revealed that these LH-RH antagonists cause mast cell degranulation, and were removed from consideration as candidates for full commercial development.
There is a need in the art for non-natural amino acids and for peptides incorporating such acids to achieve superior effects, such as, for example, improved diagnostic or disease fighting activity. None of the above references discloses the non-natural amino acids of the present invention or the advantageous properties thereof. The present invention accordingly describes positively charged non-natural amino acids, methods of making thereof, and utilization thereof in peptides.
Additional advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention provides non-natural amino acids of lysine and arginine and their derivatives (i.e., the compounds or compositions formed from performing a specified reaction) and methods for their use.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates a non-natural amino acid compound of the formula I: 
wherein
n is an integer of from 2 to 4;
R1, R2, R3 and R13 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex1 and Cxcex2 are carbon atoms and the stereochemistry at Cxcex1 and Cxcex2 is, independently, either R or S;
or the ester or salt thereof, wherein
when R1, R2, R3 and R13 are all hydrogen, then n is not 2 or 3,
when n is 2 or 3, R1 and R13 are hydrogen, and R2 and R3 are independently hydrogen or methyl with at least one of R2 and R3 being methyl, then the stereochemistry at Cxcex2 is not S, and
when n is 4, and R1, R2, R3 and R13 are all hydrogen, then the stereochemistry at Cxcex2 is not R.
The invention further relates to a non-natural amino acid compound of the formula II: 
wherein
n is an integer of from 2 to 4;
when dashed line a is not present, X and Y are independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5;
when dashed line a is present, Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R4 and R5 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof, wherein
when n is 3, dashed line a is not present, R4, X and Y are all hydrogen, and R5 is methyl, then Cxcex2 is not S,
when n is 3, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are methyl, then the stereochemistry at Cxcex2 is not R,
when dashed line a is not present, and R4, R5, X and Y are all hydrogen, then n is not 3,
when n is 4, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are the same lower branched or straight chain alkyl, then Cxcex2 is not R, and
when n is 4, dashed line a is not present, and R4, R5, X and Y are all hydrogen, then the stereochemistry at Cxcex2 is not R.
In yet another embodiment, the present invention relates to a non-natural amino acid compound of the formula III: 
wherein
n is an integer of from 2 to 4;
Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R6, R7 and R8 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof.
The invention further relates to a non-natural amino acid compound of the formula IV: 
wherein
n is an integer of from 2 to 4;
R9, R10, R11 and R12 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof.
In yet another embodiment, the invention relates to a peptide comprising the non-natural amino acid compound of formula I, II, III or IV.
In a further embodiment, the present invention provides a method for screening a peptide for an activity, comprising the steps of:
a) measuring an activity of a peptide having a selected amino acid sequence and comprising a natural amino acid;
b) measuring the same activity of a peptide having the same amino acid sequence but substituted independently in place of at least one natural amino acid, is a non-natural amino acid having the formula I, II, III or IV; and
c) comparing the measured activity of the peptides from steps a) and b) to determine whether the peptide of step b) has the activity.
In yet another embodiment, the present invention provides a method of treating or preventing in a subject a disease treated or prevented by the administration of a peptide, comprising administering to the subject a peptide having, substituted for a natural amino acid, at least one non-natural amino acid having the following formula I, II, III or IV.